The reasons organisms encode multiple Hsp70s are that it provides broader capacity to regulate abundance of Hsp70 as needs arise, or that having different Hsp70s might be a way to provide similar, but distinct functions important for carrying out specific tasks within cells in general or in specific types of cells. Earlier we constructed a yeast system to evaluate functions of Hsp70 from any source. We are using it to investigate how Hsp70s from both within and across species perform with regard to providing essential activities for cell growth and for propagation of different prions. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. Previously using this system we showed the four S. cerevisiae Hsp70s Ssa1 - Ssa4 have distinct functions that affect the propagation of different prions in different ways. These differences in the ways the prions depend on subtle distinctions in Hsp70 activities provide a sensitive way to monitor functional distinctions among the Hsp70s. For example, we identified a specific residue in the ATPase domain of the 98% identical Ssa1 and Ssa2 (A83 or G83, respectively) as solely responsible for a functional distinction between Ssa1p and Ssa2p that is critical for a vacuolar import protein degradation pathway. Our findings show that exquisitely fine differences in structure can underlie distinctions in function of nearly identical Hsp70s and that regulation of Hsp70 substrate interactions, rather than substrate binding per se, is the important parameter that determines functional distinctions. These subtle differences can lead to large-scale effects on cellular processes and might be all that is needed to direct different Hsp70s to specialized tasks in the cell. Our continued work focuses on determining if differences in functions of various Hsp70s are mediated by differences in the way they cooperate with other components of the cellular protein quality control machinery, such as co-chaperones or other major chaperones. We are also interested in learning whether such differences in Hsp70 function contribute to protection from amyloid toxicity that we see in some of our strains, and the extent to which human Hsp70s might possess such protective functions.